The FastLane Cell cDNA Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Superior sensitivity in cancer research.

In real-time two-step RT-PCR analysis of various genes important to cancer research in HeLa cells, lower CT values were achieved when cDNA was prepared with the FastLane Kit instead of a similar kit from Supplier AIV.

Successful analysis of CDC2 knockdown.

MCF-7 cells were transfected with various CDC2 siRNAs (S1, S2, or S3) or nonsilencing siRNA (NS). After 48 hours, cDNA was prepared using the FastLane Kit. Expression of CDC2 (cell cycle protein) relative to GAPDH (endogenous control) was analyzed by real-time two-step RT-PCR. The relative expression of CDC2 in untreated cells (UT) was set to 100%.

Effective elimination of genomic DNA contamination.

cDNA was prepared using the FastLane Cell cDNA Kit, with (+RT) or without (-RT) reverse transcriptase. The cDNA was quantified using a gene expression assay for β-actin. The -RT plot in indicates that no genomic DNA was detected.

Highly reproducible cDNA preparation.

cDNA was prepared from a 48-well plate containing HeLa cells using the FastLane Kit. Expression of tubulin B (structural protein) was analyzed by real-time two-step RT-PCR.

The FastLane Cell cDNA Kit uses FastLane technology to prepare cDNA directly from cultured cells in just 45 minutes. To ensure only RNA is detected in real-time RT-PCR, novel gDNA Wipeout Buffer is used to eliminate genomic DNA (see figure "Effective elimination of genomic DNA contamination"). Time and effort are saved as there is no need to design RNA-specific primers or probes.

When the FastLane Cell cDNA Kit is used together with optimized QuantiTect, QuantiFast, or Rotor-Gene Kits for real-time RT-PCR, several cell samples can be easily processed and analyzed within a few hours. This allows a snapshot of several transcripts in experiments such as validation of siRNA-mediated gene knockdown (see figure "Successful analysis of CDC2 knockdown").

The FastLane Cell cDNA Kit is part of the comprehensive range of FastLane Kits, which speed up and streamline the gene expression analysis workflow.

Procedure

With the FastLane Cell cDNA Kit, cDNA is obtained from cultured cells in just 45 minutes in a 4-step procedure: cell wash; cell lysis with integrated RNA stabilization; genomic DNA elimination; and cDNA synthesis. The synthesized cDNA is ready to use in real-time two-step RT-PCR.

The kit contains sufficient reagents for preparing cDNA from two 24-well cell-culture plates or one 48-well cell-culture plate. If working with a 96-well cell-culture plate, an additional QuantiTect Reverse Transcription Kit (50) needs to be purchased.

cDNA prepared with the FastLane Cell cDNA Kit can be analyzed by real-time two-step RT-PCR using one of the following QuantiTect Kits:

SYBR® Green detection: QuantiTect SYBR® Green PCR Kit

Probe detection: QuantiTect Probe PCR Kit

Multiplex probe detection: QuantiTect Multiplex PCR Kits

Alternatively, fast real-time two-step RT-PCR can be carried out when the FastLane Cell cDNA Kit is used in combination with a QuantiFast Kit:

SYBR® Green detection: QuantiFast SYBR® Green PCR Kit

Probe detection: QuantiFast Probe PCR Kits

Multiplex probe detection: QuantiFast Multiplex PCR Kits

Ultrafast real-time two-step RT-PCR can be achieved when cDNA prepared with the FastLane Cell cDNA Kit is analyzed on the Rotor-Gene Q cycler using one of the following dedicated Rotor-Gene Kits:

SYBR® Green detection: Rotor-Gene SYBR® Green PCR Kit

Probe detection: Rotor-Gene Probe PCR Kit

Multiplex probe detection: Rotor-Gene Multiplex PCR Kit

QuantiTect and QuantiFast Kits are compatible with various real-time cyclers, including instruments from Applied Biosystems.

Applications

The FastLane Cell cDNA Kit is suitable for experiments which require a snapshot of individual transcript levels, such as: